I started using ChIP in my analysis a short time ago and I have a lot of questions about how to normalize my data, what percentage of Input should I use and what is the best method to quantify my DNA. I received suggestions for a digital PCR, but currently we have available in my lab a nanodrop and a qubit. Which would be the most appropriate and sensitive? The main objective of my project is to determine the region in which two transcription factors interacting with the Sorghum promoter. My DNA was extracted from Arabidopsis protoplast stably transformed with my promoter, which were transfected with plasmid contructs containing the transcription factors.
ChIP (PCR)
The isolated DNA can be quantified by Real Time PCR (RT-PCR). RT-PCR typically uses TaqMan® or Sybr Green® technologies
to amplify and simultaneously quantify a targeted DNA molecule by measuring changes in fluorescence. This allows the analysis
of a specific region in multiple samples and can be quicker and more cost effective when compared to whole genome sequencing.
QBit: ChIP 1/2 to 1/5 and Input 1/10 to 1/20 for total amount
qPCR: Dilutionfactor Normalized ChIP/Input - IgG/Input CQ values.
Talking about quantification, qubit could be a little bit more sensitive. Anyway I think that nanodrop is sufficient because, in my opinion, It's more important the correct length of the fragments.
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